Brucellosis is a zoonotic disease that afflicts, depending on the region, about 5% of the livestock around the world. Although cattle, swine, sheep, goats and dogs are the usual hosts (with B. abortus, B. swis, B. ovis, B. melitensis and B. canis being the usual agents, respectively), the impact of brucellosis may be far greater as it can also infect other animals such as poultry and marine mammals, The manifestation of these bacteria in animals are usually reproductive complications (aborted fetuses, inflammed uterus or orchitis, sterility). Brucella is a bacterium that can become a facultative parasite, invading cells of the blood, bone marrow, organs and skeletal tissue. It is difficult to eliminate and relapses of infections may occur once antibiotic treatment ceases. Vaccination in animals has proven partially effective in offering protection, though these vaccines are pathogenic for other animals and humans.
Because it is a highly infective organism that causes debilitating symptoms, Brucella can persist in the environment for months under the right conditions, and as there are no effective vaccines or therapeutic recourses, it is potentially a bacterial warfare agent. There is, therefore, an urgent need to develop a means for protecting or treating people at risk.
Although antibiotics are effective in inhibiting or killing pathogens, they are less effective against pathogens that infect and then become intracellular parasites within animal or human hosts. Rather than being destroyed by white blood cells, the Brucella species, for example, thrive within these cells. Antibiotics are available that will inactivate Brucella species, but these are effective only in the test tube. In vivo, the bacterium will invade cells of the reticulo-endothelial system and become a facultative parasite, rendering it protected and difficult to eat. Antibiotics are limited in their effectiveness due to the following reasons:
only a small portion of the antibiotic may reach the infected cell due to its dilution throughout the body; PA1 some antibiotics may not be able to cross the mammalian cell membrane barrier; PA1 the antibiotic may be excreted through the urine; and, PA1 some antibiotics may become inactivated by serum or cellular enzymes. PA1 liposomes contain the antibiotic and prevent its dilution within the body or secretion in the urine; PA1 these lipid vesicles are also phagocytized and will be delivered to the site where the pathogen has sequestered; and, PA1 the liposomes are made of bio-degradable lipids and are non-toxic. Indeed, these may shield the body from the harmful side-effects of toxic antibiotics,
Current research in liposome encapsulation of antibiotics has brought in a new era in the therapy of disease. Liposomes are microscopic pockets of lipids that can be used to entrap antibiotics and to deliver these into phagocytic cells. The advantages of such a process are:
The use of liposomes as an antibiotic delivery system is described in the inventors co-pending Canadian application no. 2,101,241 (published Jan. 24, 1995) wherein liposome encapsulated ciprofloxacin was found to be more effective in the prevention and treatment of Francisella tularensis infection than the nonencapsulated antibiotic.
Further, the use of multiple doses of negatively charged liposomes as carriers of gentamicin into cells have been reported but these were only partially effective in vivo (Dees, C. et al., 1985, Vet. Immunol. Immunopathol., 8, 171-182), possibly because liposomes require phagocytosis for delivery and Brucella can invade even non-phagocytic cells (Detilleux, P. G. et al., 1990, Infect. Immun., 58, 2320-2328). Non-phagocytic cells are unlikely to engulf liposomal antibiotics and so will protect their intra-cellular parasites from these therapeutic agents. Other antibiotics will liposomes have proven effective against some strains of Brucella (e.g. B. canis and B. abortus) but less so against another strain (e.g. B. melitensis) (Hernandez-Caselles, T. et al., 1989, Am. J. Vet. Res., 50. 1486-1488). The treatment of the latter strain with antibiotics requires liposomes of a positive rather than negative charge, requires multiple treatments to be effective and although the organism may appear eliminated in mice 5 days after treatment, relapses are a possibility.
Gregoriadis, in Canadian application no. 2,109,952 (published Dec. 23, 1992), describes the use of polysaccharide coated liposomes as drug delivery agents. It is described that such polysaccharide coating is used to increase the residence time of liposomes in vivo thereby prolonging the availability of the drug. However, this reference does not address the issue of such liposomes entering non-phagocytic cells. The use of lipopolysaccharide (LPS) with liposomes has been described by Djikstra et al. (1988, J. Immunol. Meth., 114, 197-205) but the LPS was typically water-soluble and housed within the liposome rather than part of the liposome's composition.
Thus, antibiotic therapy of some diseases is very limited due to the protection offered when the facultative parasites are intracellular. Liposome encapsulation of these antibiotics enhances their effectiveness, but the indication is that there is a need for "designer" liposomes, or specific formulations of liposomes for different diseases.